Human immunodeficiency viruses (HIV) of type 1 and 2 are lentiviruses from the family of retroviruses that are believed to cause acquired immunodeficiency syndrome (AIDS). Human T lymphotropic viruses (HTLV) of type 1 and 2 are also human retroviruses and cause adult T cell leukemia, neurodegenerative diseases, and immunodeficiency. Thus, there are several types a of pathogenic human retroviruses and as used herein HIV refers to them generically. The transmission of HIV through sexual contact and during pregnancy accounts for up to 90% of AIDS cases worldwide. This transmission is initiated by the passage of HIV across the mucosal barrier of sexual organs or placenta when exposed to infectious body fluids such as semen, vaginal secretions. or blood. The remaining AIDS cases are due to the transfusion of HIV-contaminated blood, needle sharing among intravenous drug users, accidental exposure to HIV-contaminated body fluids during invasive procedures, and other situations wherein infectious virus can come into direct contact with susceptible human tissues.
Effective compounds with anti-HIV activity that could be used to prevent and/or treat AIDS are still lacking. Although initially the results with certain anti-HIV agents, e.g., azidothymidine (AZT) appeared to be promising, it has become clear that toxicity or undesirable side effects of such agents are incompatible with their antiviral activity when used at an effective pharmaceutical concentration (Bourinbaiar & Fruhstorfer, Cell Pharmacol AIDS Sci, 3:163-9, 1996). Similar concerns regarding toxicity have arisen upon use of recently introduced new drugs, such as HIV protease inhibitors. Thus, present methods of preventing and treating AIDS and HIV infection are limited and it is thus obvious that better alternative compounds devoid of toxicity and of undesirable side effects must be sought.
The present inventor has contributed to the discovery of several classes of pharmaceutical compounds which may be useful in inhibiting the replication of HIV and other viral pathogens (Bourinbaiar & Lee-Huang, Adv. Exp. Med. Biol. 374:71-89, 1995). Among them are:
Pregnancy steroid hormones, progesterone and estrogen, which were found to inhibit HIV replication in macrophages at doses smaller than can be attained by taking ordinary birth control pills. PA1 Sulphated polysaccharide, dextran sulphate, has been described as an agent suppressing cell-to-cell HIV transmission, associating for the first time the mechanism of this compound with the prevention of cell-mediated viral spread. PA1 The low doses of interferon taken orally as means to treat and prevent AIDS and HIV spread in mucosal environment. PA1 A family of heterocyclic compounds named coumarins as potent anti-HIV drugs acting as protease inhibitors. Various derivatives of coumarins are used mostly as oral anticoagulants, e.g., coumadin or warfarin. PA1 A high molecular weight factor of protein origin has been discovered in cells of placental origin as a possible candidate molecule that may help to support the fertility and prevent HIV infection during pregnancy. PA1 An oral immune modulator of bacterial origin, bestatin, as an agent with anti-HIV activity. Three clinical studies have shown that this molecule is safe and beneficial to AIDS patients. PA1 An antibiotic, gramicidin, was found to display anti-HIV, anti-herpes, and sperm-immobilizing properties which makes this compound an attractive candidate for a topical contraceptive with prophylactic capacity against sexually transmitted diseases of viral and microbial origin. PA1 N-tosyl-L-lysyl-cbloromethylketone (TLCK)--a serine protease inhibitor and HIV antagonist. PA1 The immunomodulating agent, levamisole, previously used as a deworming agent and as a drug for adjuvant therapy of malignant carcinomas. has been reported as an anti-HIV agent. PA1 Two proteins derived from medicinal plants were discovered to have anti-herpes activity. PA1 Several steroid and non-steroidal anti-inflammatory drugs such as dexamethasone, prednisone, acetylsalicylic acid (aspirin), ibuprofen, indoprofen, naproxen, nordihydroguaiaretic acid, indomethacin, etc., were tested for their activity against HIV. Some of these compounds such as prednisone have been found to be effective in delaying the progression to AIDS. PA1 Another protease inhibitor, 4'-acetarmdophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive was identified as an anti-HIV agent with activity corresponding to the levels attainable locally. PA1 Cyclosporin--an immunosuppressor used in transplant patients--was described as having anti-HIV activity. PA1 A new class of antiviral agents selected from histamine type 2 receptor antagonists such as famotidine, ranitidine, cimetidine have been identified, with activity at doses that can be attained by oral administration of a standard clinical dose. Independent clinical trials in HIV+ patients have shown beneficial effect without any adverse side-effects.
It is thus clear that the potential number of compounds that can be used in preventing and treating AIDS is quite large. However, among the many drugs that can be used today as anti-HIV agents the most promising ones are those which are naturally occurring in the human body and as a consequence are less toxic and more effective.
Human chorionic gonadotropin (hCG) is mainly known as a glycoprotein hormone secreted by the placenta and is essential for the maintenance of pregnancy. The alpha chain of hCG heterodimer is identical to alpha subunits of related glycoprotein hormones from the pituitary such as lutropin (LH), follitropin (FSH), and thyrotropin (TSH). Each hormone has a distinct beta subunit which confers receptor-binding specificities. The first 114 amino acids of beta chains of hCG and LH exhibit 86% homology as they differ by only 16 residues. However, the CTP of hCG is unique to this hormone since the beta subunit of LH lacks COOH-terminal extension. Both hormones exert their effect through a shared hCG/LH receptor--G-protein coupled single polypeptide that spans the plasma membrane seven times. As hCG-CTP does not bind to the receptor, it is believed that this portion has no biological or immunogenic activity. Due to the unique nature of the COOH-terminus of beta hCG, synthetic fragments comprising residues 109-145 have been investigated for the last 15 years as a birth control vaccine. A phase II trial of a such vaccine has recently been reported demonstrating the feasibility of this approach.
For reasons that are little understood, tumors of various origins appear to express hCG and hence, this hormone serves as an important diagnostic marker of malignancy. hCG was first proposed as an anti-tumor protein when it was observed that 7,12-dimethylbenz[a]anthracene-inducible breast carcinoma was abolished in pregnant rats or rats treated with hCG. In line with this finding, Lunardi-Iskandar et al., have recently suggested that high doses of hCG and beta hCG may be useful for the treatment of Kaposi's sarcoma by causing apoptosis in neoplastic cells. Clinical trials carried out following this report seem to lend support to this proposal.
It is now increasingly clear that hCG is not unique to pregnancy and may be responsible for a number of unrelated physiological functions. Many microorganisms and some relict invertebrates, such as horseshoe crabs, produce beta hCG-like molecules suggesting that they may play a critical role required for their survival throughout evolution. For example, Saccharomyces cerevisiae yeast appear to secrete a substantial amount of hCG-like substance which translates to the curious fact that all tested commercial beers have been found to contain this hormone. hCG is also produced in non-pregnant humans by a wide range of non-malignant cells including pituitary and T lymphocytes. hCG was found to be secreted during mixed lymphocyte reaction which results from cell-cell contact of lymphocytes. This may indicate that specialized human T lymphocytes such as cytotoxic T lymphocytes (CTL) may be responsible for the control of HIV replication by producing hCG when in contact with HIV-infected cells. Thus, hCG may be the host factor responsible for delaying the progression to AIDS and AIDS associated clinical symptoms such as cancer, opportunistic infections, wasting, and endocrine abnormalities.
The hCG/LH receptor, originally thought to be found exclusively on gonadal cells, has been now found on various cells outside of the reproductive compartment. A functional 50 kD receptor with affinity to hCG has recently been discovered on human T lymphocytes as well. The presence of this receptor on the immunocompetent cells may explain the fact that picomolar quantities of hCG can exert a strong chemotactic stimulus for T lymphocytes, monocytes, and neutrophils. Incidentally, several reports indicate that chemoattractant chemokines such as RANTES and macrophage inflammatory protein 1 (MIP-1alpha and beta) act as HIV antagonists through competition with shared cellular ligands which belong to G-linked receptors of the same family as hCG/LH receptor.
The present inventor was first to discover that pregnancy-associated glycoprotein hormone human chorionic gonadotropin (hCG) and its beta subunit can display anti-HIV activity at non-toxic doses. Dimer hCG as well as the beta hCG subunit can suppress HIV replication within the physiological dose range, suggesting that this hormone is responsible for the low rate of HIV transmission in utero. At least in an in vitro model hCG has been found to prevent cell-to-cell HIV transmission resulting from physical contact of virus-infected lymphocytes with trophoblasts--the fetal cells lining the outer layer of the placenta. Furthermore, low dose hCG injections appeared to have beneficial effect in AIDS patients by reducing the viral burden and reversing HIV infection-associated endocrine abnormalities. In a model of transgenic mice carrying defective HIV provirus, hCG was found to protect newborn mice from death and reduce HIV infection-associated skin lesions and wasting. In this model, both hCG and beta hCG have been shown to decrease HIV mRNA and HIV protein expressions. In view of such findings it is clear that hCG, which shares the same characteristics with chemoattractant chemokines, is an ubiquitous multifunctional molecule with potential in AIDS prevention and therapy. However, the active site(s) on the hCG molecule responsible for the anti-HIV effect were not identified as it was not clear to those skilled in the art whether hCG itself or contaminating molecule unrelated to hCG, found in commercial hCG preparations, was responsible for the anti-HIV effect.
Amino acids are the main components of peptides and proteins but can also be found in a free form. Amino acids are the most important class of organic substances in the cell and human body fluids. The beta hCG is a subunit specific to the most important pregnancy hormone--hCG. The N-terminal, core and C-terminal portions (CTP) of beta hCG are defined as each being approximately 50 amino acid residues long. The entire beta subunit of hCG is a 145 amino acid long peptide and may be represented by the general formula S.sub.1 KEPLRPRCR.sub.10 PINATLAVEK.sub.20 EGCPVCITVN.sub.30 TTICAGYC PT.sub.40 MTRVLQGVLP.sub.50 ALPQVVCNYR60DVRFESIRLP.sub.70 GCPRGV NPVV.sub.80 SYAVALSCQC.sub.90 ALCRRSTTDC.sub.100 GGPKDHPLTC.sub.110 DDP RFQDSSS.sub.120 SKAPPPSLPS.sub.130 PSRLPGPSDT.sub.140 PILPQ.sub.145 : Sequence I.D. No. 1.
Wherein each individual letter corresponds to a specific amino acid according to the established one-letter code nomenclature as follows: A=Alanine; R=Arginine; N=Asparagine; D=Aspartic acid; C=Cysteine; E=Glutamic acid; Q=Glutamine; G=Glycine; H=Histidine; I=Isoleucine; L=Leucine; K=Lysine; M=Methionine; F=Phenylalanine; P=Proline; S=Serine; T=Threonine; W=Tryptophan; Y=Tyrosine; V=Valine.
The beta and alpha subunits of hCG are glycoproteins, meaning that in addition to amino acids they contain various sugar molecules or oligosaccharides. Prior art indicates that gonadotropin hormones including hCG are not active if they are not glycosylated. The degree of activity of glycoprotein hormones is reduced when the glycosylation is altered.
The present invention relates to compounds selected from a group of peptides representing beta hCG comprising CTP, N-terminal end of beta hCG, or peptide fragments thereof containing at least 4 amino acid units for use as a new class of pharmaceutical agents with anti-HIV activity. The activity of these peptides can be observed even when they are not glycosylated.